1. Field of the Invention
An optical waveguide probe for assaying an analyte of interest.
2. Description of the Related Art
Fiber optic biosensors utilizing evanescent wave detection have been developed and used to detect drugs, toxins, and bacteria in food, clinical and environmental samples. The fibers were designed to use evanescent wave technology by immobilizing antibodies directly to the tip. The subsequent immunochemical reaction would bind the target antigens or organisms directly to the optical fiber and detection would occur within the evanescent wave. By utilizing the evanescent region, background from the fluorescently labeled detection antibodies was reduced to a minimum and did not interfere with detection. However, one limitation of the immunochemical assays is the regeneration of the antibodies. The antibody-antigen binding is not readily reversible and the conditions required to release the antigen result in the denaturation of the antibodies with subsequent loss of signal. Procedures have been developed for the reuse of the tapered fiber tips, but there exists a limit to the life of the sensing fiber. Disposable plastic tips have been developed to address this problem.
A second approach to the development of a reusable optical fiber biosensor utilized polystyrene microspheres for capture and isolation of the target organisms. Organisms bound to the polystyrene spheres and labeled with a fluorescently tagged secondary antibody can be detected by focusing the spheres ultrasonically into the evanescent region of a tapered fiber. The spheres used to capture the organisms could then be released and discarded leaving the sensing fiber ready for another reading. The use of a disposable to capture the target cells is very convenient and provides for the easy disposal of pathogen contaminated samples.
The parent application of the instant application, U.S. application Ser. No. 09/435,081, discloses a fiber optic magnetic-focusing immunosensor that is used to measure and detect pathogens in a sample. Paramagnetic microspheres labeled with surface-immobilized antibodies to a pathogen are mixed with the target pathogens and a fluorescent-labeled secondary antibody, for efficient one-step capture and fluorescent labeling of the cells. The microsphere-pathogen-fluorescent-label complex is placed in a cuvette in front of the fiber optic magnetic-focusing immunosensor. The fiber optic magnetic-focusing immunosensor, which is comprised of a magnetic tip, a collecting fiber and an excitation fiber, attracts the microspheres to a small concentrated spot on the side of the cuvette in front of the excitation and collecting fibers wherein the fluorescent signal is detected and measured.
The present invention comprises an improved magnetic-focusing immunosensor for detecting and measuring a pathogen in a sample.
Broadly the invention comprises magnetic focusing of paramagnetic microspheres with a waveguide probe. Microspheres with immobilized antibodies interact throughout the analyte containing the target antigens, which, in turn, capture fluorescent-labeled antibodies in a standard sandwich assay. The bound antigen/antibody/fluorescent antibody complexes are magnetically attracted to the waveguide probe which contains the sensing volume of the excitation and collection fibers, while the uncaptured labeled fluorescent antibodies remain in bulk solution thereby reducing background fluorescence.
The invention comprises a hollow waveguide probe comprising first and a second tubes each having a middle portion and converging at a proximal end and a distal end. At the proximal end, the tubes converge to zero degrees. A magnet is positioned in the proximal end. An excitation fiber is in communication with the middle portion of the first tube which function as a transmission conduit. Excitation light is injected into the first tube and is guided to the proximal end where it illuminates the inner wall of a cuvette where fluorescent complexes are attracted by the magnet. The associated fluorescent signal is collected by the second tube and is guided to the distal end of the probe wherein the signal is detected and measured.
In a preferred embodiment of the invention a primary antibody specific for the antigen (pathogen) to be detected is coated on a magnetic bead and a secondary antibody is conjugated to a marker. A food sample is prepared and added to the medium. If the expected pathogen, such as Salmonella Typhimurium is present, the pathogen binds to both antibodies forming a magnetic complex. The magnetic complex is attracted to the proximal end of the probe. The proximal end is polished to form a glass receiving aperture having a diameter which is approximately 2xc3x97 the wall thickness of the first tube. The fluorescent signal received from the second tube is dettected. The signal can be detected by a fiber optic spectrometer in communication with a computer or a PIN detector in communication with an optical power meter.
In another aspect of the invention, a sample from the blood of mammals, fish or fowl is prepared and added to a medium comprised of primary antibodies specific for the anitgens (pathogens) to be detected whereby the primary antibodies are coated on a magnetic bead and a secondary antibody conjugated to a marker. The waveguide probe of the present invention could then be utilized as described above.
In yet another aspect of the invention, the waveguide probe is a portable unit that can be used for field testing.